Summary Glioblastoma (GBM) is among the deadliest human cancers with a median survival of 15 to 19 months. Over 90% of patients treated with the current standard of care, concomitant temozolomide and radiotherapy, show tumor progression while on treatment. Consequently, recurrence is inevitable and the optimal management re- mains unclear. There is an urgent need to develop strategies for improving treatment options for GBM. GBM is among the most comprehensively genomically characterized tumors, which has confirmed extensive inter- tumoral heterogeneity. These studies have identified genetic lesions that provide a rationale for targeted treat- ments, but to date, the accumulated genetic characterization of GBMs has failed to significantly impact clinical practice and patient outcomes. In addition, extensive intratumoral heterogeneity in GBM is documented as bears functional relevance, influencing response to therapy. This includes varied metabolic adaptations that have been documented in GBM. The clear dependency of GBM on altered metabolic programs and an evolving under- standing of metabolic functional heterogeneity and adaptation in cancer prompted an unbiased screen for gene products that feed essential metabolic pathways in GBM. This identified Medium-chain Acyl-CoA Dehydrogen- ase (MCAD) as candidate feeder of GBM. The long-term goal of this research is identify novel therapeutic targets for the treatment of GBM. The overall objective is to evaluate the oncogenic mechanism of MCAD in GBM to determine whether this dependency represents a therapeutic opportunity, either directly through targeted inhibi- tion of MCAD, or indirectly by targeting the cancer-relevant pathways it regulates. The central hypothesis is that the essential activities of MCAD in GBM influence lipid metabolism, energy production, and/or epigenetic repro- gramming. The rationale for focusing on MCAD is based on the strong emergence of ACADM and other acyl- CoA dehydrogenases from the screening that uniquely employed low-passage, patient-derived glioma spheres (GSCs) in vivo to interrogate an shRNA library targeting 330 metabolism genes, convincing in vitro and in vivo validation of the dependence of GBM models on MCAD, and a very high frequency of elevated MCAD expression in human disease. The specific aims are to (i) elucidate the MCAD mechanism of action, (ii) evaluate the effect of MCAD on epigenetic reprogramming of cells, and (iii) evaluate tumor impact and tolerability of acute MCAD expression. This grant is significant because a deep understanding of the cancer-essential mechanism(s) of MCAD in GBM may illuminate a new molecule or pathway to target that may improve outcomes for patients with this deadly disease. It is anticipated that the research may be of high translational relevance if it is determined that effects of MCAD targeting may synergize with standard-of-care and/or targeted therapies that perturb DNA repair. The research is innovative because targeting lipid degradation enzymes represents a novel approach for a potential cancer therapeutic.